2.1 Ribozymes
A ribozyme is a catalytic RNA molecule that cleaves RNA in a sequence specific manner. The use of ribozymes as potential gene regulators in mammalian cells and antiviral agents has been suggested, but subject to serious questions regarding technical feasibility. For example, there are differences regarding how ribozymes can be introduced to target cells or how they can be directed to the same subcellular compartments as their target RNAs. Other questions concern the effects of target RNA secondary structure on ribozyme activity. The art has not been successful in definitively answering any of these questions.
Furthermore, because riboozymes are a form of antisense technology, the problems encountered in applying antisense technology to disease treatment are also encountered in the use of ribozyme technology (Haseloff, J., and W. L. Gerlach, 1988, Nature 334(6183):585-91; Sullivan, S. M., 1994, J. Invest Dermatol. 103(5 Supl):858-895). For example, it has been shown that the expression of antisense RNA in transgenic mice did not invariably lead to a reduction in target RNA molecules, and when reduction in target RNA molecules did occur, it was not predictably paralleled by a reduction in protein. Even when protein levels were reduced sometimes no biological effect was detected (Whitton, J. Lindsay “Antisense Treatment of Viral Infection” Adv. in Virus Res. Vol. 44, 1994).
The experience in the art suggests that it is also not clear whether ribozymes work best when free or when associated with only short non-specific flanking sequences, or when embedded in unrelated larger RNA molecule (Whitton, 1994, supra). At present, sufficient data are not available, either in vitro or in cell culture to allow systematic comparison of the transactivities of free ribozymes with their embedded equivalents.
There have been some studies that focus on the potential use of ribozyme technology in the treatment of cancer. In these studies, ribozymes have been directed against both c-fos and c-ras oncogenes in cell culture, and these showed some suppression of the malignant potential of transfected cells when transplanted into mice. Nevertheless, these ribozymes specifically target an oncogene.
There has been no suggestion in the literature that tissue-specific cancers or other tissue-specific disease can be treated by delivering to that tissue a ribozyme having a tissue-specific promoter, and that it is targeted to an RNA that is essential for cell survival. The invention provides such a ribozyme capable of treating tissue-specific cancers and other tissue-specific diseases.
2.2 Promoters
Furthermore, endogenous delivery of a ribozyme under the control of a tissue-specific or other promoter can be complicated by “leakiness”, where low levels of transcription occur in extraneous tissues. This could present a considerable therapeutic problem, depending upon the cellular target chosen. The present invention compensates for this problem by targeting a cellular target which is associated with high levels of product (such as, RNA polymerase I produces large amounts of cellular ribosomal RNA). Thus, in the event promoter leakiness occurs in unintended tissues, it is not likely that cell death would occur. This choice, therefore, provides a needed level of safety, and targeting of pol I would be applicable to many selected tissues using other promoters.
2.3 Delivery
Another common problem in gene therapy is the difficulty in delivering the ribozyme to the correct tissue. The present invention avoids this difficulty by targeting the ribozyme to non-cellular RNAs in cells to which ribozyme constructs can be efficiently delivered. IV liposome delivery will be effective for treatment of HBV hepatitis. Intravenous and/or extracorporeal treatment will effectively delivery construct to erythrocytes for treatment of malarial infection. And topical (with or without iv) administration will effectively deliver ribozyme construct to cervical epithelium in dysplastic/precancerous/cancerous HPV 16 cervical lesions. This latter example is of extreme importance for treatment of dysplastic/carcinoma in situ lesions diagnosed via abnormal Pap smears. A second advantageous facet of the non-cellular target ribozymes of the present invention is that even if promoter leakiness and/or extraneous delivery and/or expression of the ribozyme occurs in unintended cells, the ribozymes should not cleave the cellular RNAs.
2.4 Antimicrobial Agents
Infectious diseases sicken or kill millions of people each year. Numerous antimicrobial therapies have been designed to target one or several infectious agents. These therapies show varying degrees of success in eradicating infection. However, the failure of many of these therapies to target specific infectious agents has lead to overuse or inappropriate use of the therapies, which in turn has lead to the development of drug resistant microbes. The development of drug resistance in many infectious agents has reduced the efficacy and increased the risk of using the traditional antimicrobial therapies.
While ribozymes have been known and studied for several years, they have not been used in the treatment of bacterial infections. There are many reasons for this. A key technical concern in the use of ribozymes as antimicrobial agents is that the ribozyme must be taken up and expressed by the targeted microbe so that the ribozyme(s) can cleave the targeted RNA(s) inside the microorganism. A second important concern is the tight coupling of transcription and translation in microorganisms which can complicate efficient cleavage of targeted bacterial RNAs. These concerns are addressed by the present invention.